Abstract

Epigenetic control of gene expression is vital for normal development and
differentiation of cells, and is also important in the development of disease. In
particular, there is a strong association between hallmark epigenetic changes and
cancer – namely, genome wide hypomethylation, gene specific hypermethylation
and characteristic histone modification. Almost all studies of cancer epigenetics
to date have been conducted in malignant tissues or already transformed cell
lines, and therefore do not take into account epigenetic changes occurring during
the process of transformation. Our lab has developed a line of primary
mesenchymal stem cells (MSC; thought to be the origin of various types of
sarcoma) in which five oncogenic steps towards a fully transformed state are
sequentially introduced including: human telomerase, necessary to extend the life
span of MSC in culture, genes to inactivate the p53 and pRb tumour suppressor
genes and genes to activate the oncogenes c-Myc and Ras. I hypothesized that
hallmark epigenetic changes take place in this step-wise model of transformation,
and aimed to investigate genome wide hypomethylation and the activity of the
polycomb repressive 2 (PRC2) complex in this system. Utilizing the PCR based
technique MethyLight, I show that transformed MSC are hypomethylated
compared to parental MSC, with this decrease in methylation occurring on the
introduction of oncogenic H-Ras in the final step. I also show that this
hypomethylation is a gradual event following H-Ras expression, and
transformation can take place in the absence of hypomethylation. I demonstrate
that the three core components of the PRC2 complex are up-regulated during
step-wise transformation and that PRC2 target genes are down-regulated. Finally,
I show that MSC are able to be transformed when the PRC2 components EZH2
and SUZ12 are knocked down before the final oncogenic hit. These studies show
that hallmark epigenetic changes occur during step-wise transformation and
suggest that tumour-associated epigenetic changes occur following genetic
aberrations. This model is valuable and relevant to further explore the
mechanisms behind epigenetic alterations in cancer.